The immune system has evolved to mount defensive responses against invading pathogens, and at the same time, to protect the human body from self-destruction. T cells are critical in the induction of the immune tolerance, and breakdown of such tolerance results in autoimmune diseases such as type I diabetes. The peripheral T cell tolerance involves several mechanisms including deletion, ignorance, anergy, and the generation of T regulatory cells. Our long-term goal is to understand protein ubiquitination in the regulation of the immune response. Ubiquitin (Ub)-dependent proteolysis has been implicated in a variety of cellular processes, and abnormalities in the Ub system have been shown to cause pathological responses, including malignant transformation, and several genetic diseases. The E3 Ub liases are the critical components that provide specificity to the Ub systemby direct interaction with particular substrates. Itch, a novel E3 Ub ligase, is absent in the non-agouti-lethal 18H mice, or Itchy mice. These mice develop immunological and inflammatory diseases in the lung and stomach, hyperplasia in lymphoid and hematopoietic tissues, and constant itching in the skin, suggesting that Itch is involved in the regulation of immune responses. Itch contains an N-terminal protein kinase C-related C2 domain, four WW domains and C-terminal HECT ligase domain. At present, the biological pathways affected by Itch mutation remain largely unclear. Particularly, recent studies have pointed out a critical role of E3 ligases including Itch and Cbl-b in the T cell unresponsiveness or anergy. Based on our preliminary studies, we propose that, first, Itch targets its substrates for ubiquitination through the interaction of its protein-binding domains and subsequently regulates signaling transduction pathways via the ubiquitination of these target proteins; second, the deficiency of Itch affects T cell function via T cell tolerance induction; and third, Itch and Cbl-b have functional synergy in regulating T cell activation. Our proposed plan is to test these hypotheses by examining Itch-mediated ubiquitination of its substrates, Itch-involved regulation of intracellular signal transduction in Itch deficient primary T cells and transiently transfected cells. The study on Itch is likely to shed light on the biological function of E3 ligases in the induction of T cell tolerance, and may provide insights for therapeutic intervention in immunological diseases.